Post-translational modifications of histone proteins play a fundamental role in the organization of chromatin and in the regulation of gene transcription. Our long-term objective is to elucidate how histone ubiquitinases and histone methyltransferases regulate gene expression and control cell growth. Two such enzymes, which are highly conserved, are the histone H2B ubiquitinase Rad6 and the histone H3 lysine 4 (K4) methyltransferase Set1. We have recently demonstrated that Rad6-mediated histone H2B K123 ubiquitination is essential for histone H3 K4 methylation in yeast, thereby linking these two forms of histone modification to gene activation. Rad6 is an E2 ubiquitin-conjugating enzyme whose functions are evolutionary conserved from yeast to human. Importantly, the human RAD6 homolog (hHR6B) was found to be over-expressed in breast cancer cell lines and in breast carcinomas. Moreover, over-expression of exogenous hHRGB resulted in chromosome instability and transformation of human epithelial cells. Many human homologs of the H3 K4 methyltransferase SET1 (e.g., MLL, Mixed Lineage Leukemia) are also mutated or disrupted in a variety of human cancers. Collectively, these findings establish a strong link between histone ubiquitination and methylation with cancer. Given the highly conserved functions of Rad6 in regulating H2B ubiquitination and H3 methylation, it is therefore of paramount importance to understand the roles of Rad6 in mediating these histone modifications, and ultimately, controlling gene expression and cell growth. Our overall hypothesis is that through H2B ubiquitination and subsequent H3 K4 methylation, Rad6 regulates the transcription of specific genes that are important for controlling cell growth. Therefore, it is imperative to understand the basic molecular mechanisms of action of Rad6-mediated H2B ubiquitination in establishing histone H3 methylation, and thereby, gain insight into the role of Rad6 in controlling gene transcription. Using budding yeast as a model organism, we plan to use a combination of biochemistry and genetics to dissect the function of Rad6 in these processes. To achieve this, three Specific Aims are proposed. Aim 1. Identify and characterize factors that mediate the functions of Bre1 and Rad6. Aim 2. Define the roles of phosphorylation and acetylation on Rad6. Aim 3. Determine the molecular mechanisms of action of Rad6-mediated H2B ubiquitination in establishing histone H3 methylation.